Virion preparations

Estimate and record the concentration of HBV DNA in a small aliquot of the HBV virion preparation by slot-blot hybridization in parallel with known standards. 

More recently, the use of liquid chromatography and tandem MS (LC/MS/MS) has also eased purification and recovery methods. For instance, Vamum and colleagues utilized gel-free two-dimensional capillary LC/MS/MS and Fourier transform ion cyclotron resonance MS to identily and determine the relative abundances of viral and cellular proteins in purified HCMV virions and dense bodies. Analysis of the proteins Irom purified HCMV virion preparations has indicated that the particle contains significantly more viral proteins than previously known. They identified more than 71 HCMV-encoded proteins and 70 host cellular proteins in HCMV virions, which included cellular structural proteins, enzymes, and chaperones [6]. Another study using LC/MS/MS for the adenovirus type 5 pro-teome found a total of 11 protein species from 154 peptides, at a sensitivity of 10 copies per virus and a detection limit of 70 finol for two proteins [36]. 

Kreuter and Speiser [77] developed a dispersion polymerization producing adjuvant nanospheres of polymethylmethacrylate) (PMMA). The monomer is dissolved in phosphate buffered saline and initiated by gamma radiation in the presence and absence of influenza virions. These systems showed enhanced adjuvant effect over aluminum hydroxide and prolonged antibody response. PMMA particles could be distinguished by TEM studies and the particle size was reported elsewhere to be 130 nm by photon correlation spectroscopy [75], The particle size could be reduced, producing monodisperse particles by inclusion of protective colloids, such as proteins or casein [40], Poly(methylmethacrylate) nanoparticles are also prepared... 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

Although this procedure is necessary for production of samples of pure virions it is usually unnecessary if a viral preparation is only required for subsequent infections. In such circumstances the cells should be harvested aseptically and processed to step e, omitting the DNase and RNase. The debris from the disrupted cells is pelleted at 15,000g for 30 min and the supernatant used as a source of virus. It should be tested for bacterial contamination with brain-heart infusion broth and Saboraud fluid medium (Appendix 4). The virus should be stored at -70°C at about 1010 p.f.u./ml. 

Virion templates of TMV were also used in combination with different synthetic routes for CdS, PbS, and Fe oxide nanoparticles. Nanoparticle-virion tubules were prepared by reacting a buffered solution of TMV in CdCl2 (pH 7) or TMV in Pb(N03)2 (pH 5) with H2S gas. The formation of metal sulfide nanoparticles occurred over 6 hours as observed by a uniform coating of CdS and PbS nanocrystals on the TMV surface from TEM analysis. Selected area electron diffraction of the mineralized products indicated a zinc blende crystal stracture for CdS particles and a rock salt structure for single domain PbS nanocrystals. The iron oxide nanoparticles were mineralized by the TMV templates by the oxidative hydrolysis of an Fe VFe acidic solution with NaOH. Consequently, a mineral coating of irregular ferrihydrite particles grew on the surface to a thickness of 2 nm. 

Recently, Young et al. have designed a CCMV virion cage with an anionic interface that favorably binds Fe and Fe ions and then subsequently controls the oxidative hydrolysis to size-constrained iron oxide minerals. The anionic cage was prepared by genetically replacing the 9 basic residues... 

Start the polymerase reaction by adding 50-pL aliquots of HBV virion or core preparation to each tube. 

Fig. 2.4 Flowchart of the steps for rAAV production as described in Protocol 1. HEK293 cells are expanded, transfected, and harvested at 60h posttransfection. The cells are lysed, and loaded onto either a cesium chloride gradient or an iodixanol gradient to separate infectious virions from empty capsids. Virus is purified using either heparin affinity chromatography or ion-exchange chromatography. Virus preparations are formulated and concentrated, and characterized...

GPC proved of great value in the preparation of viral proteins, because the fractions obtained could be subjected to further analysis. A study of viral proteins is of interest from the point of view of not only the virion structure but also the immunological properties. 

To quantitate virus particle concentrations on a molal basis, which is appropriate for thermodynamic analysis of adsorption data, we measured virus particle concentrations spectroscopically 13) with concentrated, highly purified preparations, using an absorbance constant of 1.32 X 10 virions per absorbance unit-cm at 260 nm (8). Particle to PFU ratios varied from 355 to 18621. 

In MCMV infected macrophages and fibroblast, M78 is mainly localized inside the cells in vacuole like structures which colocalizes with markers for medial and peripheral Golgi bodies. Furthermore, M78 is present in virus preparations, and is as a possible virion envelope protein, presumably delivered to newly infected target cells. M78 has been proposed to be involved in modulating the intracellular milieu and regulating the transcription of selected immediate early viral mRNAs. ... 

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